The goal of this project is to elucidate the mechanism by which Cortistatin A inhibits HIV-1 transcription. Human immunodeficiency virus type I (HIV-1) is the causative agent of AIDS (Acquired Immunodeficiency Syndrome). HIV anti-retroviral therapy is based on the administration of drugs in combination, in order to minimize development of mutations that can confer single-drug resistance to the virus. The viral protein Tat, a potent activator of HIV gene expression, is a potential antiviral target. We have discovered that Cortistatin A is a promising anti-Tat drug candidate. Cortistatin A is a steroidal alkaloid isolated from the marine sponge Corticium simplex. We have found that didehydro-Cortistatin A (CA for short), is a very potent inhibitor of Tat- activated transcription of the HIV-1 provirus. CA is extremely efficient at reducing viral output from acutely infected cells as well as from chronically infected cultured cells or freshly isolated peripheral blood lymphocytes (PBLs). The half maximal effective concentration (EC50) of CA for inhibiting HIV in chronic infected cells is less than 0.1 nM and the half maximal cytotoxic concentration (CC50) for CA in cultured cell lines or freshly isolated PBLs is more than 20 5M, which confers to CA a very promising therapeutic index. We have identified an interaction between CA and Tat and with the DNA-dependent protein kinase (DNA-PK) catalytic subunit. Upon HIV infection, DNA-PK tightly associates with Tat to promote phosphorylation of the transcription factor Sp1, which results in increased HIV-1 transcription initiation, via three Sp1 binding sites in the core of the HIV-1 promoter. Our hypothesis is that CA might repress HIV replication in two ways: by inhibiting Tat-TAR interaction, thereby inhibiting elongation by RNA Polymerase I (Pol I) from the HIV promoter; and/or by inhibiting DNA-PK activation of the transcription factor Sp1 and initiation/elongation of transcription by Pol II. Both routes would result in repression of HIV-1 transcriptional activation. Our specific aims include: Aim 1.Determine whether CA inhibits HIV-1 Tat-TAR interaction in vitro and in vivo and if CA inhibits transcription elongation from the viral promoter. Assess whether CA inhibits Tat mediated DNA-PK phosphorylation of Sp1. Aim 2. In-depth assessment of CA toxicity in vivo. Aim 3. Determine CA effect on HIV-1 replication in humanized mice models. We will thus elucidate the mechanism by which CA inhibits HIV-1 transcription and evaluate its potential to inhibit HIV-1 replication in humanized mice. In order to generate a backup compound we will evaluate additional analogs generated during the course of this project. These analogs will be subsequently tested in the humanized mouse model.